A key feature of the neuronal changes accompanying long-term memory storage is the formation of new synaptic connections. Despite the association of morphological changes with different forms of learning, little is known about the mechanisms that underlie this synaptic structural plasticity. To study these mechanisms, we have exploited the cellular specificity of a simple behavioral system - the gill-withdrawal reflex of the marine mollusc Aplysia californica. Long-term memory for sensitization of this reflex is associated with the growth of new synaptic connections between identified sensory neurons and their follower cells. These changes can be reconstituted in dissociated cell culture by the repeated presentation of serotonin (5-HT), a facilitating neurotransmitter normally released by sensitizing stimuli in the animal. We now propose to combine video-enhanced imaging of the living terminal, thin-section electron microscopy, molecular probes, and gene transfer methods with the experimental accessibility of sensorimotor cocultures to determine the cellular and molecular events responsible for the induction and maintenance of this learning-related synaptic growth and synapse formation. Toward that end we plan five types of experiments, which are designed to address the following: (l) contribution of growth to the early and late stages of long-term memory, (2) role of immediate-early genes in induction of the structural change, (3) pre- and (4) postsynaptic regulation of the, structural change, and (5) requirement of protein synthesis for the structural change. In this fashion we hope to gain additional insights into the mechanisms that contribute to these structural changes as well as to clarify how the molecular and structural changes associated with learning are related to those of synapse formation during development. The approaches we have developed and the model system we plan to use provide the required specificity to address these problems directly and should increase our understanding of the signaling pathways that are involved in the flow of information from the surface membrane to the genome during learning. Since structural changes are such a prominent feature of the long-term process, principles derived from this reductionist approach may be applicable to more complex systems and ultimately to human memory.